WO2016175351A1 - Rice-derived osiaa6 gene for increasing drought stress resistance of plant and use thereof - Google Patents

Abstract

Disclosed are: a method for controlling the resistance of a plant to environmental stress, comprising a step of transforming plant cells with a recombinant vector including a gene coding for a rice-derived Oryza sativa Aux/IAA 6 (OsIAA6) protein; a method for producing a transgenic plant body of which the resistance of a plant to environmental stress is controlled, comprising a step of transforming plant cells with the recombinant vector including a gene coding for the OsIAA6 protein; a transgenic plant body produced by the method and of which the resistance to environmental stress is controlled; and seeds thereof.

Description

Rice-derived OSISIA6 gene and its use to increase drought stress tolerance in plants

The present invention relates to a rice-derived OsIAA6 gene and its use for increasing drought stress resistance of plants, and more particularly, to a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein. A method of controlling resistance to environmental stress of a plant, comprising the step of transforming a cell, transforming plant cells with a recombinant vector comprising a gene encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein The present invention relates to a method for producing a transgenic plant having a controlled resistance to environmental stress of a plant, a transgenic plant having a controlled resistance to environmental stress produced by the method, and a seed thereof.

Drought stress is one of the most serious factors affecting crop production worldwide. Exposure of plants to drought conditions leads to the induction of many stress-related genes, the products of which are thought to function as cellular protective agents against stress-induced damage. The expression of stress-related genes is usually regulated by specific transcription factors, which account for about 6% of the genome total gene number in rice and Arabidopsis.

The AUX / IAA (auxin / indoleacetic acid) gene encodes a family of proteins whose expression is tightly regulated by the plant hormone auxin. The plant hormone auxin is involved in a variety of processes such as cell division, cell expansion and differentiation, embryonic patterning, growth control of vasculature or other tissues, root and side roots or stem meristem. AUX / IAA proteins generally have four conserved amino acid sequence motifs (domains I, II, III and IV) and nuclear position signal sequences. Domains I and II play a role in degrading AUX / IAA proteins by recognizing auxin signals as degradation domains. Domains III and IV are protein-protein interaction dimerization domains known to form homodimers with AUX / IAA proteins or heterodimers with ARF proteins. AUX / IAA proteins inhibit ARF activity as negative regulators of auxin response factors (ARFs) that regulate expression of auxin-responsive genes. In the auxin activated state, the AUX / IAA protein is ubiquitized through interaction with the auxin-SCFTIR1 complex and then degraded by proteasome action. An overview of the role and activity of AUX / IAA proteins has been reported by Reed (Trends in Plant Science 6, 420-425, 2001). Structure and expression analysis of early auxin-responsive AUX / IAA gene families in rice (Oryza sativa Aux / IAA 6) was recently reported by Jain et al. (Funct Integr Genomics. 2006 Jan; 6 (1): 47-59). A total of 25 ARF genes and 31 Aux / IAA genes are present in rice, and the various processes of planting control the development of the whole plant by auxin. The purpose of this study is to isolate OsIAA6 genes that increase expression in drought, salt, and low temperature abiotic stress treatment, and analyze the function of drought resistance of the genes.

Meanwhile, Korean Patent No. 0742194 discloses a method of increasing environmental stress resistance of a plant using environmental stress resistance control genes, and Korean Patent No. 1427180 discloses rice which increases drought stress resistance of plants. Derived OsCTR1 gene and its use 'is disclosed, but there is no known about the OsIAA6 gene derived from rice and its use to increase drought stress resistance of plants as in the present invention.

The present invention has been derived by the above-described requirements, the present inventors transformed the plant with a recombinant vector comprising a rice-derived OsIAA6 protein coding gene and the recombinant vector to produce a rice transgenic plant overexpressing the OsIAA6 gene, By confirming that the OsIAA6 overexpressed transgenic rice plants increased drought stress resistance compared to wild type plants, the present invention was completed.

In order to solve the above problems, the present invention provides a plant-resistant resistance to environmental stress, comprising the step of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein It provides a way to adjust.

In addition, the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein; And

It provides a method for producing a transformed plant is controlled resistance to environmental stress comprising the step of regenerating the plant from the transformed plant cells.

In addition, the present invention provides a transgenic plant and its seed having a controlled resistance to environmental stress produced by the method.

According to the present invention, plants overexpressing the genes encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein have increased stress resistance compared to wild type under drought stress conditions. Therefore, the gene encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein of the present invention can be usefully used in the development of transgenic plants with increased resistance to drought stress. Could be.

1 is a result of confirming the change in the expression level of OsIAA6 gene during treatment of drought, salt or low temperature in rice plants.

Figure 2 is a result of confirming the change in the expression level of OsIAA6 gene when the plant was treated with NAA (naphthalene-1-acetic acid), an analogue of Auxin, a growth hormone of rice.

3 is OsIAA6 transgenic plants: The results confirm the expression level change of the gene in OsIAA6 (PGD1 OsIAA6).

4 is OsIAA6 transgenic plants: a result of comparing the drought stress tolerance of (PGD1 OsIAA6) and wild type plants, (A) is a photograph of the drought stress treatment and the plant state of after the water resupply (rewatering), (B ) This is a graph analyzing the change of Fv / Fm value according to drought stress treatment.

In order to achieve the object of the present invention, the present invention is directed to the environmental stress of plants comprising the step of transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein Provides a way to control tolerance.

Method according to an embodiment of the present invention is characterized by increasing the plant's resistance to environmental stress by overexpressing a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein in plant cells However, it is not limited thereto.

In the present invention, "environmental stress" refers to an external factor that lowers the growth or productivity of a plant and is largely classified into a biological stress and an abiotic stress. Biological stresses typically include pathogens, and abiotic stresses include high concentrations of salt, drought (dry), low temperature, high temperature and oxidative stress. "Environmental stress tolerance" refers to a trait in which a decrease in growth or a decrease in productivity of a plant caused by environmental stress is suppressed or delayed.

In the method according to an embodiment of the present invention, the environmental stress may be a drought stress, but is not limited thereto.

OsIAA6 according to the invention The range of proteins includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of such proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means activity that modulates the environmental stress tolerance of a plant.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and intracellularly reintroduced by artificial means.

In the present invention, the OsIAA6 gene sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host.

Expression vectors comprising the OsIAA6 gene sequence of the present invention and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.

Preferred examples of recombinant vectors of the invention are Ti-plasmid vectors capable of transferring part of themselves, the so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance gene, aadA gene, and the like, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, Clp promoter. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the recombinant vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens) Terminator of the octopine gene, and the rrnB1 / B2 terminator of Escherichia coli, but are not limited thereto. With regard to the need for terminators, such regions are generally known to increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

When transforming the vector of the present invention into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2) , 3T3, RIN and MDCK cell lines) and plant cells and the like can be used. The host cell is preferably a plant cell.

The method of transporting the vector of the present invention into the host cell may be performed by injecting the vector into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, or the like. can do.

In addition, the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein; And it provides a method for producing a transformed plant is controlled resistance to environmental stress comprising the step of regenerating the plant from the transformed plant cells.

Method according to an embodiment of the present invention is characterized by increasing the resistance to environmental stress of the plant by overexpressing a recombinant vector comprising a rice-derived OsIAA6 gene in plant cells, but is not limited thereto.

Preferably, the OsIAA6 protein may be composed of the amino acid sequence of SEQ ID NO: 2.

In the method according to an embodiment of the present invention, the environmental stress may be a drought stress, but is not limited thereto.

The method of the present invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformation may be mediated by, for example, Agrobacterium tumefiaciens ( Agrobacterium tumefiaciens ). The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

Transformed plant cells should be re-differentiated into whole plants. Techniques for regeneration of mature plants from callus or protoplast cultures are well known in the art for many different species.

In addition, the present invention provides a transgenic plant and its seed having a controlled resistance to environmental stress produced by the method.

Preferably, the transgenic plant and its seeds are transgenic plants and their seeds with increased resistance to environmental stress.

The plant is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, gatchi, watermelon, melon, cucumber, Dicotyledonous plants such as pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, peas, or monocotyledonous plants, such as rice, barley, wheat, rye, corn, sugarcane, oats, and onions, preferably monocotyledonous plants, and more. Preferably it may be a rice plant, but is not limited thereto.

The present invention also provides a composition for regulating environmental stress resistance of plants, comprising a gene encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient. The composition contains a gene encoding an OsIAA6 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient, the plant is transformed into a plant by transforming the gene or a recombinant vector containing the gene into an environmental stress tolerance, preferably drought. It can increase stress tolerance.

The present invention also provides the use of a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein consisting of the amino acid sequence of SEQ ID NO: 2 to regulate the environmental stress resistance of plants. The gene encoding the OsIAA6 protein can preferably be used to increase the drought stress resistance of the plant.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Materials and methods

Plasmid Construction and Transformation of Rice

OsIAA6 (AK068600) and full-length cDNA are the template of total RNA extracted from rice ('ilmi' cultivar), and the OsIAA6 forward (5'-caccatggaagaagggtccaacaaaa-3 '; SEQ ID NO: 3) and the OsIAA6 reverse (5'-ttagaccctagcagtagctcca -3') SEQ ID NO: 4) using a primer. Amplified respective full-length cDNA by using the Gateway system (Invitrogen, USA) was inserted into the vector p700 with a PGD1 promoter. PGD1: OsIAA6 vectors were introduced into plants via the Agrobacterium mediated co-culture method (Jang et al., 1999, Mol. Breeding 5: 453-461).

Stress and auxin analog (NAA; naphthalene-1-acetic acid) treatment

To analyze the trend of expression of the OsIAA6 gene in response to abiotic stress, nontransformed (NT, 'Ilmi' cultivar) rice plants were grown for 4 weeks in soil in a plastic house. Four-week-old transgenic plants were air dried for three hours for drought stress treatment. In addition, the whole plant was transferred to 200 mM sodium chloride solution for salt stress for 24 hours, and the whole plant was placed in a 4 ° C. low temperature chamber for 24 hours for low temperature stress treatment.

For transgenic analogues to plants, non-transformed (NT, 'Ilmi' varieties) rice plants were grown in a chamber for 6 days, and the plants were transferred to MS medium containing 100 uM NAA for 24 hours.

Real-Time Quantitative RT-PCR

After removing and freezing the leaf tissue of abiotic stress and auxin analog (NAA; naphthalene-1-acetic acid) treated plants, the frozen leaf and root tissues are pulverized with liquid nitrogen and Qiagen RNeasy plant mini kit (Qiagen, USA) ) Was used to extract total RNA. 1 μg of total RNA was used for first strand cDNA synthesis using the Superscript II cDNA synthesis kit (Invitrogen, USA). To analyze gene expression levels, Mx3000p real-time PCR instrument and Platinum SYBR  Quantitative PCR was performed using the Green qPCR SuperMix-UDG system (Invitrogen, USA). Rice ubiquitin transcript was used as a standardization index, and OsIAA6 gene specific primers were prepared at 3 'UTR and 3' exon sites. Primers used for quantitative RT-PCR are shown in Table 1 below.

Table 1

Gene name		Sequence information (5 '→ 3')
OsIAA6	Forward direction	AGC ACC AGT GGT TGG TTG GCC (SEQ ID NO: 5)
	Reverse	CCC AAC CAG CAT CCT GTC TCC T (SEQ ID NO: 6)
OsUbi 1	Forward direction	ATG GAG CTG CTG CTG TTC TA (SEQ ID NO: 7)
	Reverse	TTC TTC CAT GCT GCT CTA CC (SEQ ID NO: 8)

Phenotypic Analysis of Drought Stress

PGD1: OsIAA6 transgenic plants were grown with soil for 4 weeks in the soil and then treated with drought stress by removing water from the soil. After 5 days of drought stress treatment, water was replenished and visual symptoms of drought stress in PGD1: OsIAA6 transgenic plants and non-transformed plants were photographed and observed.

Pulse-Amplitude Modulation (PAM) Experiment

Three lines of PGD1: OsIAA6 transgenic plants were grown in the soil for two weeks with non-transgenic plants, and then leaves were collected from each plant and allowed to acclimate for 10 minutes. For PAM analysis, drought conditions were treated in a growth chamber in a time-lapse fashion, and at each indicated time, chlorophyll fluorescence emitted from the upper surface of the leaves was extracted using a pulse modulated fluorophotometer (mini-PAM, Walz, Germany). The Fv / Fm value was measured by sensing. Fv / Fm values indicate activity of light system II.

Example 1 Under Abiotic Stress Conditions OsIAA6 Analysis of Gene Expression Trend

To determine how the expression trend of OsIAA6 gene changes in response to abiotic stress, including drought stress, drought, high salt, and low temperature stress were treated in non-transformed rice. As a result, it was confirmed that the expression of OsIAA6 gene was increased 6 times in drought stress, 4 times in high salt stress, and 2.3 times in low temperature stress compared to the control group (FIG. 1).

Example 2. By Auxin Treatment OsIAA6 Gene expression analysis

Auxins are one of the major hormones in plants that regulate their growth and development. The auxin reaction regulates the expression of ARF genes through the signaling system by accurately recognizing the amount of auxin in the plant. The Aux / IAA protein is a negative regulator of the ARF protein. Fine tune the In order to check whether OsIAA6 gene, which is significantly increased in abiotic stress conditions among 31 Aux / IAA genes in rice, reacted with auxin, NAA, an auxin analogue, was treated with 100 uM for 24 hours in wild type rice grown 6 days. . As a result, the OsIAA6 gene showed about 2.7-fold increase in expression compared to the untreated control (FIG. 2). As a result, it was confirmed that OsIAA6 gene, which is significantly increased under abiotic stress conditions of rice, reacts auxin.

Example 3. OsIAA6 Drought Resistance Verification by Gene Overexpression

In Example 1 described above, it was confirmed that OsIAA6 gene was strongly expressed under drought stress conditions. In order to confirm the potential function on the drought resistance of OsIAA6 gene, OsIAA6 overexpressed transgenic rice plants were prepared, and the expression pattern of OsIAA6 gene was confirmed in the overexpression (FIG. 3). In addition, drought stress was treated to identify drought resistance in overexpression of 5 lines. After 3 days of drought stress treatment, the wild type transgenic plants showed mild symptoms of drought induced damage such as leaf curling and wilting. With the increase of drought stress treatment time, the symptoms of wild type transgenic plants became severe, and on the 5th day of drought treatment, there was a strong symptom of drought induction. The leaves of the non-transformed plants were all dried, and the plants did not recover from the drought stress-induced damage even when water was resupplied (FIG. 4A). However, OsIAA6 overexpressing transgenic plants showed strong resistance to drought stress. OsIAA6 overexpressed transgenic plants showed less leaf damage than wild-type nontransgenic plants even after 3 days of drought stress treatment. On the 5th day of drought treatment, both non-transformers and OsIAA6 overexpressed transgenic plants suffered from drought severe leaves. However, the stems of the plants were firmly maintained compared to the non-transformed plants. Recovery from stress-induced injury. The above results show that OsIAA6 gene can enhance the resistance to drought stress.

To confirm the function of OsIAA6 in drought resistance, pulse-amplitude modulation (PAM) tests were performed using three independent lines of OsIAA6 overexpressing transgenic plants. The Fv / Fm values of the PAM assay were used to observe the effects of abiotic stress in plants, and the nontransformed plants showed a strong decrease in Fv / Fm values by drought stress treatment. The Fv / Fm value was 0.8 when no stress was treated in wild type transgenic plants, but the Fv / Fm value began to decrease after 1 hour of drought stress treatment, and the decrease accelerated with increasing stress treatment time. (FIG. 4B, wild type). On the other hand, unlike non-transformed plants, OsIAA6 overexpressed transgenic plants showed delayed decrease in Fv / Fm values due to drought stress. Three OsIAA6 overexpressing transgenic plants maintained higher values than wild type transgenic plants until 2 hours of drought stress treatment. These results supported that overexpression of OsIAA6 enhanced plant resistance to drought.

Claims (12)

1. A method of controlling plant resistance to environmental stress, comprising the step of transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein.
2. The method of claim 1, wherein the gene encoding the OsIAA6 (Oryza sativa Aux / IAA 6) protein is overexpressed in plant cells, thereby increasing the plant's resistance to environmental stress.
3. According to claim 1, wherein the OsIAA6 protein is characterized in that consisting of the amino acid sequence of SEQ ID NO: 2.
4. The method of claim 1, wherein the environmental stress is a drought stress.
5. Transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein; And

   Method for producing a transformed plant is controlled resistance to environmental stress comprising the step of regenerating the plant from the transformed plant cells.
6. The method according to claim 5, wherein the gene encoding the OsIAA6 (Oryza sativa Aux / IAA 6) protein is overexpressed in plant cells to prepare a transgenic plant having increased resistance to environmental stress.
7. The method of claim 5, wherein the OsIAA6 protein consists of the amino acid sequence of SEQ ID NO: 2. 7.
8. 6. The method of claim 5, wherein said environmental stress is a drought stress.
9. A transgenic plant with controlled resistance to environmental stress produced by the method of claim 5.
10. The plant of claim 9, wherein the plant has increased resistance to environmental stress.
11. 10. The transgenic plant of claim 9, wherein the plant is a monocotyledonous or dicotyledonous plant.
12. Transformed seeds of the transgenic plant according to claim 9.

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